1. Field of the Invention
The present invention relates generally to the fields of molecular virology and vaccinology. More specifically, the present invention relates to the attenuation of negative stranded RNA viruses by rearrangement of their genes and uses thereof.
2. Description of the Related Art
Live attenuated viruses capable of replicating to generate protective humoral as well as cell mediated immune responses without producing disease manifestations have proven effective vaccines against viruses such as smallpox, yellow fever and poliomyelitis. The strategy for attenuation, however, has been empirical in most cases and not reproducible for general use. An additional consideration in the case of RNA viruses is that the high error rate of RNA dependent RNA polymerases, their lack of proof reading and the quasi-species nature of RNA virus populations (Domingo et al, 1996), make the use of live attenuated viruses for this large group of medically significant pathogens problematic. This is especially true if the vaccine virus is based on a limited number of single base changes as mutation to virulence is a potential problem. For example, only a few back mutations can restore virulence to the Sabin poliovirus type 3 vaccine strain (Wimmer et al., 1993).
The non-segmented negative strand RNA viruses of the family Mononegavirales possess an elegantly simple means of controlling the expression of their genes. The linear, single-stranded RNA genomes of this family encode five to ten genes, the order of which is highly conserved among all members. The prototype virus of this family is the Rhabdovirus, vesicular stomatitis virus (VSV). Transcription of the viral genome is carried out by the virus encoded RNA dependent RNA polymerase. There is a single entry site on the linear genome for the RNA polymerase, yet the mRNAs of the virus are not produced in equimolar amounts.
Available evidence indicates that the linear order of the genes on the genome controls the levels of expression of individual genes. Transcription initiates at the single polymerase entry site at the 3' terminus of the genome and is obligatorily processive (Ball and White, 1976). The level of expression of the individual genes as monocistronic mRNAs is controlled by the dissociation, approximately 30% of the time, of the polymerase at each intergenic junction, as it traverses the genome in the 3' to 5' direction (Iverson and Rose). This mechanism of transcription results in sequentially decreasing amounts of the transcripts of each gene as a function of the distance of the gene from the 3' terminus of the genome. Correspondingly, gene products needed in stoichiometric amounts to support replication, such as the nucleocapsid (N) protein, are encoded at or near the 3' terminus in all cases and expressed in the highest molar amounts (Villarreal e t al., Ball and White). Gene products needed in enzymatic amounts, such as the RNA polymerase are encoded most distal from the 3' end. In all cases, the polymerase gene is the 5'-most gene, expressed in the lowest amount. Precise molar ratios of the proteins are required for optimal replication. For successful replication, proteins must be expressed in molar ratios that approximate those expressed normally from the genome (Pattnaik and Wertz, 1990).
Viruses of the family Mononegavirales do not undergo homologous genetic recombination (Pringle, 1987). Thus, other than defective interfering particles, which lack portions of the genome, variants of these viruses having the entire complement of genes in a rearranged format have not been observed in nature.
The prior art is deficient in the lack of effective means of attenuating negative stranded RNA viruses by rearrangement of their genes and uses of such attenuated viruses for vaccines. The present invention fulfills this longstanding need and desire in the art.